1. Field of the Invention
This invention relates to a method of detecting a target neoplastic nucleic acid in histologic tissue external to a primary neoplasm, and reagents useful therein.
2. Description of Related Art
An increasing body of evidence implicates somatic mutations as causally important in the induction of human cancers. These somatic mutations may accumulate in the genomes of previously normal cells, some of which may then demonstrate the phenotypes associated with malignant growth. Such oncogenic mutations may include a number of different types of alterations in DNA structure, including deletions, translocations and single nucleotide alterations. The latter, also known as point mutations, may frequently intervene in carcinogenesis, since a variety of mutagenic chemicals induce such mutations. In addition, such mutations may occur spontaneously as a result of mistakes in DNA replication.
Advances in recombinant DNA technology have led to the discovery of normal cellular genes (proto-oncogenes and tumor suppressor genes) that control growth, development, and differentiation. Under certain circumstances, the regulation of these genes is altered, causing normal cells to assume neoplastic growth behavior. There are over 40 known proto-oncogenes and suppressor genes to date, which fall into various categories depending on their functional characteristics. These include, (1) growth factors and growth factor receptors, (2) messengers of intracellular signal transduction pathways, for example, between the cytoplasm and the nucleus, and (3) regulatory proteins influencing gene expression and DNA replication.
Point mutations have been directly implicated in the causation of many human tumors. Some tumors carry oncogenes of the ras gene family, which differ from their normal cellular counterpart proto-oncogenes by the presence of a point mutation at one of a limited number of sites in these genes. Similarly, point mutations in critical regions of tumor suppressor genes, such as p53, are often detected in tumor cells. Mutation of the p53 suppressor gene is the most common alteration seen in epithelial tumors and, indeed, in all human tumors (Hollstein, M. et al., Science 253:49-53, 1991).
When a tumor suppressor gene, such as p53, becomes mutated, cell proliferation accelerates in the absence of the suppressor. On the other hand, mutations in proto-oncogenes that transform them to active oncogenes, such as a mutant ras oncogene, produces cell proliferation caused by presence of the mutant gene itself. These mutations represent qualitative changes in the tumor cell genome that distinguish these cells from normal cells and provide a basis for diagnosis of the genetic origin of a tumor under study.
Identification of the mutations that have created active oncogenes may provide important diagnostic and prognostic clues for tumor development. For example, a number of mutations have been found to alter the 12th codon of the ras oncogenes, causing replacement of a normally present glycine by any of a number of alternative amino acid residues. Such amino acid substitutions create a potent transforming allele. Thus, the presence of a particular nucleotide substitution may be a strong determinant of the behavior of the tumor cell (e.g., its rate of growth, invasiveness, etc.). As a result, nucleotide hybridization probes of oncogene mutations have promise as diagnostic reagents in clinical oncology.
Head and neck squamous carcinoma, commonly associated with mutant p53, kills over 11,000 Americans each year, yet little is known concerning the genetic events involved in progression of these malignancies. A number of neoplasms found in the gastrointestinal tract, especially colorectal cancer, are better understood and commonly associated with oncogene mutations. Colorectal cancer is the third most common malignancy in the world, with 570,000 new cases expected each year. Treatment of all cancers depends on the tumor stage as determined by clinical evaluation and surgical resection. The standard technique for assessing the spread of a tumor is surgical resection of a primary tumor followed by careful review using light microscopy of surgical margins and other tissue, including lymph nodes. Under existing procedure, the adjacent tissue is stained by standard techniques and assessed under light microscopy for the presence of tumor cells. Accurate therapeutic staging assesses the extent of tumor spread locally as well as the presence of regional metastases in more distant sites, such as lymph nodes. Accurate histopathologic assessment is critical since it provides important prognostic indicators that determine the probability of survival for a given patient following surgical resection of the primary tumor.
Despite many years of research and billions of dollars in expenditures the long term survival of patients with malignancies remains disappointedly low, even where no tumor cells were detected in the tumor margins or more distant tissues. This inability to more accurately stage such patients might be due to the limitation inherent is the standard histopathologic methodology which is based upon visual observation and morphologic assessment under light microscopy of adjacent tissue and regional lymph nodes. Thus, a method which uses a more precise technique capable of determining spread of the disease at an earlier stage might provide a more accurate indication of the extent of tumor metastases into adjacent and regional tissues. The present invention provides such a method.